Once the tissue is fixed, it is further processed for observation with the microscope. This usually means making sections for staining and later observation. As a general rule, samples are hardened before sections are obtained. The thinner we want the sections the more hardened has to be the sample. Tissues are already hardened by fixation, but not so much, so that sections thicker than 20-30 µm are difficult to obtain. The fixative provides enough hardening if thick sections (between 30 and 200 µm) are what we need. Devices like vibratome can get thick sections directly from fixated tissues, and it is used when a very good molecular preservation is required. However, thinner sections are usually needed, so the sample must be hardened. There are two common procedures to do this: freezing and embedding.
Freezing of previously fixated tissues provides hardening that allows obtaining sections with thickness ranging from about 50 µm to nanometers. Several sectioning apparatus are used: freezing microtome for thick sections (larger than 20 µm), cryostat for thin sections (from 5 µm to about 20 µm) and ultracryotome for ultrathin sections (nanometers). To prevent damages caused by freezing, like formation of crystals of water that can make holes in the tissues, samples immersed in cryoprotecting solutions. Sucrose (30%) is the most common cryoprotector, but dimethyl sulfoxide, glycerol, ethylene glycol are also used. They are selected depending on the type and size of the sample and procedure to be later performed. Another important parameter is the freezing speed. The quicker the sample is frozen the smaller the water crystal, so it minimizes the tissue damages. For example, freezing in liquid nitrogen is frequently used.
Embedding is a widely used method for hardening samples. Embedding substances in liquid state are infiltrated in the samples and after a period of cooling or polymerization the embedding substance, and therefore the sample, gets solid without altering the morphological or molecular features of tissues. In this way, depending on the embedding substance, very thin section can be obtained without breaking or spoiling the tissue. In addition, embedding is a good method for preserving samples during long periods of time. A number of embedding substances are available to get specific section thickness and for performing particular processing methods of the sections. For light microscopy observation, paraffin is the most common embedding substance. Celoidine is another embedding medium for light microscopy. For electron microscopy, epoxy and acrylic resins are the most used embedding substances. The majority of the embedding media are not hydrosoluble, i.e., they are not miscible with water. It means that the water of the sample should be exchanged with water insoluble liquids. If some water is remaining in the sample, the embedding process does not properly reach all the sample parts, so that we will have sections and tissues with poor quality.